Differential Properties of Two Putative Nuclear Localization Sequences Found in the Carboxyl-Terminus of Human IFN- γ

Abstract

Interferon-γ (IFN-γ), a protein that uses the Jak-Stat pathway for signal transduction, translocates rapidly to the nucleus in cells treated extracellularly with the cytokine. A nuclear localization sequence (NLS) has been identified and characterized in the C-terminus of IFN-γ. Both human and murine IFN-γ contain this NLS. We show in this report that human IFN-γ (HuIFN-γ) contains a second NLS at an upstream site, as determined in standard import assays using digitonin-permeabilized HeLa cells. The primary sequence, analogous with the NLS sequence identified in murine IFN-γ, representing amino acids 122-132 of HuIFN-γ was capable of mediating the nuclear import of the autofluorescent protein allophycocyanin(APC) in an energy-dependent manner. The second sequence, representing amino acids 78-92 of HuIFN-γ, was also capable of mediating the nuclear import of APC in an energy-dependent manner but to a greatly reduced extent. The nuclear import of both sequences conjugated to APC was strongly blocked by competition with unconjugated HuIFN-γ(122-132). Competition by the sequence HuIFN-γ(78-92) effectively blocked the import of APC-conjugated HuIFN-γ(78-92) but, at the same concentration, was not capable of inhibiting the nuclear import of APC-conjugated HuIFN-γ(122-132), suggesting that HuIFN-γ(78-92) was a less efficient NLS than HuIFN-γ(122-132). This is consistent with >90% loss of antiviral activity of HuIFN-γ lacking the downstream NLS in 122-132. The nuclear import of APC-conjugated HuIFN-γ(122-132) was inhibited by a peptide containing the prototypical polybasic NLS of the SV40 T NLS, which suggests that the same Ran/importin cellular machinery is used in both cases.